GnRH Pulse Generation in Rodents: Time to Terminate the Role of Dynorphin?

The pulsatile release of gonadotrophin-releasing hormone (GnRH) from neurons in the hypothalamus is necessary to initiate and maintain pituitary gonadotrophin release and gonadal function. In the 1970s, the arcuate nucleus of the hypothalamus (ARC) was proposed to contain an oscillatory cell population that acts as the GnRH pulse generator (1); however, the phenotype of this population remained elusive for several decades. Studies in sheep in the late 1980s and 1990s identified that endogenous opioid peptide (EOP) signaling participated in steroid hormone negative feedback control of GnRH pulse generation (2). Furthermore, the role of EOPs as an inhibitor of pulse frequency was replicated in human patients (3), with EOP antagonism increasing the frequency of pulsatile luteinizing hormone (LH) release, an indirect measure of GnRH release. Notably, the antagonism of EOP in the ovine ARC prolongs the length of a GnRH pulse, providing critical evidence for a role in pulse termination (4).

The 2000s revealed the essential role of the neuropeptides kisspeptin and neurokinin B (NKB) in fertility and as stimulators of GnRH/LH release (2). Multiple groups using rodent and ovine models reported that the ARC contains a population of cells that co-express kisspeptin and NKB with the EOP dynorphin (2). For ease, these cells were termed KNDy neurons (pronounced kan-dee—a nod toward the discovery of kisspeptin in Hershey, Pennsylvania, and the naming of the peptide after Hershey's Kisses). KNDy neurons also express the kappa opioid receptor (KOR), which has a high affinity for dynorphin, and multiple receptors for neurokinin B (2). In vitro recordings in mouse brain slices confirmed that NKB stimulated and dynorphin inhibited KNDy cell activity, with the effects of these peptides blocked by antagonists against their receptors (5). This compelling collection of pharmacological, anatomical, and in vitro electrophysiological evidence bought forth the “KNDy hypothesis,” in which reciprocal connections between KNDy cells permit NKB and dynorphin to auto-stimulate and auto-inhibit the population, respectively, in an oscillatory manner. This episodic activation of KNDy cells may lead to pulses of kisspeptin release, which acts through its receptor at GnRH neurons to elicit the GnRH pulse. Central to this hypothesis is the role of KOR in the termination of KNDy cell activity and GnRH secretion. However, the necessity of KOR signaling within KNDy cells for GnRH pulse generation and resultant fertility has not yet been demonstrated.

To test the in vivo role of KOR in the KNDy hypothesis, Coutinho and colleagues (6) recently developed a mouse line with the conditional knockout of KOR from kisspeptin cells (Kiss KORKO mice) and conducted a robust analysis of the reproductive and neuroendocrine profiles of males and females. Surprisingly, although KOR antagonism in rodents advances the onset of puberty and results in an associated increase in LH pulse frequency (7), the removal of KOR from kisspeptin cells in this study did not alter the age of puberty onset. Furthermore, contrary to the KNDy hypothesis, LH pulses persisted in adult Kiss KORKO mice with unaltered amplitude and frequency of release and no apparent change in duration. As KOR is also expressed in kisspeptin neurons in the rostral periventricular nucleus of the third ventricle (RP3V) (8), strongly considered to respond to estradiol positive feedback and drive the LH surge that triggers ovulation, female mice were also subject to a surge-induction protocol. They observed no difference in the ability of knockout mice to mount an LH surge in response to high estradiol levels. In line with an unaltered neuroendocrine profile, downstream reproductive measures were also unaffected by the knockout, with no changes in estrous cyclicity and fecundity detected. Validation of the knockout using RNAscope-based in situ hybridization confirmed the loss of KOR mRNA from kisspeptin cells in the RP3V and ARC. Together, these observations suggest that, at least in mice, KOR-KNDy signaling is not obligatory for reproductive neuroendocrine development.

The results of this study raise numerous possibilities regarding the involvement of KOR in GnRH pulse generation in rodents. Potentially, dynorphin termination of KNDy cell activity may occur indirectly through a KOR-expressing non-KNDy population that has efferent and afferent connectivity with KNDy cells. Alternatively, the rodent pulse generator may contain a yet unidentified non-KOR mechanism that inhibits KNDy cell activity. This is supported by pharmacological data in which nor-binaltorphimine, a KOR-specific antagonist, increases LH pulse frequency in ruminants and human subjects but has little effect when used in rodents (2). This theory is consistent with the findings by Coutinho et al, as it suggests that KNDy cells are capable of being inhibited by exogenous stimulation with dynorphin or a KOR agonist but that endogenous KOR activation at KNDy cells is not obligatory for GnRH pulse termination.

It should be noted that the knockout of KOR from kisspeptin cells in embryonic or early postnatal development may lead to compensation to maintain reproductive capacity. However, this possibility supports the theory that a separate mechanism to KOR-KNDy signaling is sufficient to terminate KNDy cell activity and GnRH pulses. Although technically challenging, future experiments ablating KOR expression specifically from KNDy cells in adulthood will be required to eliminate the possibility of developmental compensation that masks the role of KOR-KNDy signaling on GnRH pulse generation and subsequent fertility measures.

In conclusion, the study by Coutinho et al (6). provides in vivo evidence that KOR expression in KNDy cells is unnecessary for neuroendocrine reproductive development in mice. These findings highlight the need to expand the KNDy hypothesis to include non-KNDy and non-KOR pathways that terminate the GnRH pulse. Whether this unidentified “stop” signal is present in non-rodent species and is necessary to terminate GnRH pulses remains to be determined.

Abbreviations

ARC

arcuate nucleus of the hypothalamus

EOP

endogenous opioid peptide

GnRH

gonadotropin-releasing hormone

KNDy neurons

kisspeptin neurokinin B and dynorphin neurons

KOR

kappa opioid receptor

LH

luteinizing hormone

NKB

neurokinin B

RP3V

rostral periventricular nucleus of the third ventricle

Funding

This publication was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under Award Number R00HD096120 to A.M.M.

Disclosure

The author have nothing to disclose.

Data Availability

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.